The optimization of cell-SELEX based aptamer selection through masking DNA, PCR and non-SELEX

Aptamers are short oligonucleotide sequences that are capable of binding with high affinity and specificity to a wide variety of targets. The selection of aptamers from a random oligonucleotide library, by cell-SELEX, has lead to their in vivo application in both research and clinical settings. Cell-SELEX continues to be hindered by the non-specific binding of selection sequences to the complex cell surface as well as low specificity in the PCR amplification of DNA. In this work the cell-SELEX procedure was optimized to improve the efficiency of aptamer selection using three modifications: masking DNA characterization, non-SELEX selection and touchdown PCR. An established masking DNA model was tested against the MCF-7 and 4T1 cell lines and was shown to be capable of determining the masking DNA concentration needed for aptamer selection. The traditional SELEX approach was combined with a non-SELEX aptamer selection protocol to reduce the concentration and heterogeneity of sequences collected after the first round of selection thus leading to more accurate PCR amplification and a decrease in byproduct formation. Lastly, touchdown PCR was used to successfully eliminate the amplification of genomic DNA reducing the formation of genomic DNA byproducts.